DNA encoding and 18 KD CDK6 inhibiting protein

ABSTRACT

A nucleic acid encoding a CDK inhibiting protein, particularly a CDK6 inhibiting protein, is selected from the group consisting of: (a) DNA having the nucleotide sequence given herein as SEQ ID NO:1 (which encodes the protein having the amino acid sequence given herein as SEQ ID NO:2), and which are refered to as p18 INK6  ; (b) nucleic acids which hybridize to DNA of (a) above and which encode a CDK inhibiting protein; and (c) nucleic acids which differs from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and which encodes a CDK inhibiting protein encoded by a DNA of (a) or (b) above. Constructs containing such DNA, cells containing such constructs, proteins encoded by such DNA, antibodies which bind thereto, antisense oligonucleotides corresponding to such DNA, and methods of using the same are also disclosed.

FIELD OF THE INVENTION

The present invention relates to cyclin-dependent kinase (CDK)inhibiting proteins in general, and particularly relates to DNA encodingan 18 Kilodalton inhibitor of CDK6.

BACKGROUND OF THE INVENTION

The cyclin-dependent kinases, or "CDKs", are a family of proteinsinvolved in cell-cycle regulation. The CDKs are only active as kinaseswhen they associate with other proteins known as cyclins, on which theyare dependent. The manner by which the CDKs control cell cycleregulation has, however, only recently begun to be explained.

Y. Xiong et al., Cell 71, 505-514 (1992) showed that, in vivo in normalhuman fibroblasts, there exists a quaternary complex of cyclin D, p21,CDK, and proliferating cell nuclear antigen (or "PCNA"). See also H.Zhang et al., Molec. Biol. Cell 4, 897 (1993); Y. Xiong et al., Genes &Development 7, 1572 (1993). These results indicated that in addition tothe cyclin activation and subunit phosphorylation, the activity of CDKsmay be controlled by a number of small proteins (e.g.,p21^(WAF1/cip1/sdi1) and p16^(INK4)) that physically interact withcyclins, CDKs or cyclin-CDK complexes.

Y. Xiong et al., Nature 366, 701-704 (Dec. 16, 1993), describes thecloning of p21 and shows that p21 is an inhibitor of cyclin kinases (p21had previously been cloned and described as a senescent cell-derivedinhibitor, or "sdi", by J. R. Smith, U.S. Pat. No. 5,302,706). This workand the work of others (see El-Deiry et al., Cell 75, 817-825 (1993)further shows that p21 is upregulated by the upregulation of the tumorsupressor protein p53. By showing that p21 is under the control of p53,this work indicates that the p21 serves as a critical link between thetumor supressor protein p53 and the CDK cell cycle control mechanisms.

A. Kamb et al., Science 264, 436-440 (Apr. 15, 1994) and T. Noborl etal., Nature 368, 753-756 (Apr. 21, 1994) describe the isolation of themultiple tumor supressor 1 and 2 DNA, (or "MTS1" and "MTS2"), whichencode the CDK4 inhibitor p16 (previously identified in M. Serrano etal., Nature 366, 704-707 (Dec. 16, 1993)). It is suggested that both p16and p21 are expected to antagonize entry into the S phase of the cellcycle, and that in vitro p16 appears more specific than p21.

SUMMARY OF THE INVENTION

A first aspect of the present invention is an isolated nucleic acidencoding a CDK inhibiting protein, particularly a CDK6 inhibitingprotein. The nucleic acid may be selected from the group consisting of:

(a) DNA having the nucleotide sequence given herein as SEQ ID NO:1(which encodes the protein having the amino acid sequence given hereinas SEQ ID NO:2), and which we refer to as p18^(INK6) ;

(b) nucleic acids which hybridize to DNA of (a) above (e.g., understringent conditions) and which encode a CDK inhibiting protein; and

(c) nucleic acids which differs from the DNA of (a) or (b) above due tothe degeneracy of the genetic code, and which encodes a CDK inhibitingprotein encoded by a DNA of (a) or (b) above.

A second aspect of the present invention is a nucleic acid constructhaving a promoter and a heterologous nucleic acid operably linked tosaid promoter, wherein said heterologous nucleic acid is a nucleic acidas given above, along with cells containing such nucleic acid constructs(e.g., wherein the cell is one which expresses the encoded protein).

A third aspect of the present invention is a protein encoded by anucleic acid as given above. Such proteins may be isolated and/orpurified in accordance with known techniques.

A fourth aspect of the present invention is an antibody (e.g., apolyclonal antibody, a monoclonal antibody) which specifically binds toa protein as given above.

A fifth aspect of the present invention is an antisense oligonucleotidecomplementary to a nucleic acid as given above and having a lengthsufficient to hybridize thereto under physiological conditions, alongwith DNA encoding such an antisense oligonucleotide, and a nucleic acidconstruct having a promoter and a heterologous nucleic acid operablylinked to said promoter, wherein the heterologous nucleic acid is a DNAencoding such an antisense oligonucleotide.

A sixth aspect of the present invention is a method of inhibiting DNAsynthesis in a human cell (e.g., a tumor cell) which comprises providingto the cell a protein as given above in an amount effective to inhibitDNA synthesis therein. The cell may be provided in any suitable form,such as in in vitro culture. The providing step may be carried out byany suitable means, such as by delivering the protein into the cell orby delivering into the cell a nucleic acid encoding the protein andwhich expresses the protein in the cell.

A seventh aspect of the present invention is a method for increasing DNAsynthesis in a cell, wherein DNA synthesis in the cell is inhibited, byproviding to the cell an antisense oligonucleotide as given above in anamount effective to increase DNA synthesis in said cell. The cell may bea skin cell, such as a cell present in wound or burn tissue. Again theproviding step may be carried out by any suitable means, such as bydelivering the antisense oligonucleotide into the cell, or by deliveringinto the cell a nucleic acid encoding an antisense oligonucleotide asgiven above and which transcribes the antisense oligonucleotide in thecell.

The foregoing and other objects and aspects of the present invention areexplained in detail in the specification set forth below.

DETAILED DESCRIPTION OF THE INVENTION

Amino acid sequences disclosed herein are presented in the amino tocarboxy direction, from left to right. The amino and carboxy groups arenot presented in the sequence. Nucleotide sequences are presented hereinby single strand only, in the 5' to 3' direction, from left to right.Nucleotides and amino acids are represented herein in the mannerrecommended by the IUPAC-IUB Biochemical Nomenclature Commission, or(for amino acids) by three letter code, in accordance with 37 CFR §1.822and established usage. See, e.g., PatentIn User Manual, 99-102 (November1990)(U.S. Patent and Trademark Office).

A. DNA Sequences

DNAs of the present invention include those coding for proteinshomologous to, and having essentially the same biological properties as,the proteins disclosed herein, and particularly the DNA disclosed hereinas SEQ ID NO:1 and encoding the protein given herein SEQ NO:2. Thisdefinition is intended to encompass natural allelic variations therein.Thus, isolated DNA or cloned genes of the present invention can be ofany species of origin, including mouse, rat, rabbit, cat, porcine, andhuman, but are preferably of mammalian origin. Thus, DNAs whichhybridize to DNA disclosed herein as SEQ ID NO:1 (or fragments orderivatives thereof which serve as hybridization probes as discussedbelow) and which code on expression for a protein of the presentinvention (e.g., a protein according to SEQ ID NO:2) are also an aspectof this invention (subject to the proviso that the MTS1 and MTS2 DNAsdisclosed in A. Kamb et al., Science 264, 436 (1994) are excluded fromthis definition of the instant invention). Conditions which will permitother DNAs which code on expression for a protein of the presentinvention to hybridize to the DNA of SEQ ID NO:1 disclosed herein can bedetermined in accordance with known techniques. For example,hybridization of such sequences may be carried out under conditions ofreduced stringency, medium stringency or even stringent conditions(e.g., conditions represented by a wash stringency of 35-40% Formamidewith 5× Denhardt's solution, 0.5% SDS and 1× SSPE at 37° C.; conditionsrepresented by a wash stringency of 40-45% Formamide with 5× Denhardt'ssolution, 0.5% SDS, and 1× SSPE at 42° C.; and conditions represented bya wash stringency of 50% Formamide with 5× Denhardt's solution, 0.5% SDSand 1× SSPE at 42° C., respectively) to DNA of SEQ ID NO:1 disclosedherein in a standard hybridization assay. See, e.g., J. Sambrook et al.,Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring HarborLaboratory)). In general, sequences which code for proteins of thepresent invention and which hybridize to the DNA of SEQ ID NO:1disclosed herein will be at least 75% homologous, 85% homologous, andeven 95% homologous or more with SEQ ID NO:1. Further, DNAs which codefor proteins of the present invention, or DNAs which hybridize to thatof SEQ ID NO:1, but which differ in codon sequence from SEQ ID NO:1 dueto the degeneracy of the genetic code, are also an aspect of thisinvention. The degeneracy of the genetic code, which allows differentnucleic acid sequences to code for the same protein or peptide, is wellknown in the literature. See, e.g., U.S. Pat. No. 4,757,006 to Toole etal. at Col. 2, Table 1.

Knowledge of the nucleotide sequence as disclosed herein in SEQ ID NO:1can be used to generate hybridization probes which specifically bind tothe DNA of the present invention or to mRNA to determine the presence ofamplification or overexpression of the proteins of the presentinvention. For example, oligonucleotide probes that are homologous toboth DNA of SEQ ID NO:1 and to MTS1 or MTS2 DNA encoding p16 asdescribed in M. Serrano et al., Nature 366, 704-707 (Dec. 16, 1993) andA. Kamb, Science 264, 436 (1994), may be used to locate homologous DNAsof the same family in the same species or other species. Examples ofsuch probes are the probes having the sequences given herein as SEQ IDNO:3 and SEQ ID NO:4. The hybridization probes may be cDNA fragments oroligonucleotides, and may be labelled with a detectable group asdiscussed hereinbelow. Pairs of probes which will serve as PCR primersfor the DNA sequences of the present invention, or portions thereof, maybe used in accordance with the process described in U.S. Pat. Nos.4,683,202 and 4,683,195 to Mullis (applicant specifically intends thatthe disclosures of all U.S. Patent references disclosed herein beincorporated herein by reference).

B. Genetic Engineering Techniques

The production of cloned genes, recombinant DNA, vectors, transformedhost cells, proteins and protein fragments by genetic engineering iswell known. See, e.g., U.S. Pat. No. 4,761,371 to Bell et al. at Col. 6line 3 to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clark et al. atCol. 4 line 38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 to Schilling atCol. 3 line 26 to Col. 14 line 12; and U.S. Pat. No. 4,879,224 toWallher at Col. 6 line 8 to Col. 8 line 59. (Applicant specificallyintends that the disclosure of all patent references cited herein beincorporated herein in their entirety by reference).

A vector is a replicable DNA construct. Vectors are used herein eitherto amplify DNA encoding the proteins of the present invention or toexpress the proteins of the present invention. An expression vector is areplicable DNA construct in which a DNA sequence encoding the proteinsof the present invention is operably linked to suitable controlsequences capable of effecting the expression of proteins of the presentinvention in a suitable host. The need for such control sequences willvary depending upon the host selected and the transformation methodchosen. Generally, control sequences include a transcriptional promoter,an optional operator sequence to control transcription, a sequenceencoding suitable mRNA ribosomal binding sites, and sequences whichcontrol the termination of transcription and translation. Amplificationvectors do not require expression control domains. All that is needed isthe ability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants.

Vectors comprise plasmids, viruses (e.g., adenovirus, cytomegalovirus),phage, retroviruses and integratable DNA fragments (i.e., fragmentsintegratable into the host genome by recombination). The vectorreplicates and functions independently of the host genome, or may, insome instances, integrate into the genome itself. Expression vectorsshould contain a promoter and RNA binding sites which are operablylinked to the gene to be expressed and are operable in the hostorganism.

DNA regions are operably linked or operably associated when they arefunctionally related to each other. For example, a promoter is operablylinked to a coding sequence if it controls the transcription of thesequence; a ribosome binding site is operably linked to a codingsequence if it is positioned so as to permit translation. Generally,operably linked means contiguous and, in the case of leader sequences,contiguous and in reading phase.

Transformed host cells are cells which have been transformed ortransfected with vectors containing DNA coding for proteins of thepresent invention, constructed using recombinant DNA techniques.Transformed host cells ordinarily express protein, but host cellstransformed for purposes of cloning or amplifying DNA coding for theproteins of the present invention need not express protein.

Suitable host cells include prokaryotes, yeast cells, or highereukaryotic organism cells. Prokaryote host cells include gram negativeor gram positive organisms, for example Escherichia coli (E. coli) orBacilli. Higher eukaryotic cells include established cell lines ofmammalian origin as described below. Exemplary host cells are E. coliW3110 (ATCC 27,325), E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294(ATCC 31,446). A broad variety of suitable prokaryotic and microbialvectors are available. E. coli is typically transformed using pBR322.See Bolivar et al., Gene 2, 95 (1977). Promoters most commonly used inrecombinant microbial expression vectors include the beta-lactamase(penicillinase) and lactose promoter systems (Chang et al., Nature 275,615 (1978); and Goeddel et al., Nature 281, 544 (1979)), a tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980)and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al.,Proc. Natl. Acad. Sci. USA 80, 21 (1983)). The promoter andShine-Dalgarno sequence (for prokaryotic host expression) are operablylinked to the DNA of the present invention, i.e., they are positioned soas to promote transcription of the messenger RNA from the DNA.

Expression vectors should contain a promoter which is recognized by thehost organism. This generally means a promoter obtained from theintended host. Promoters most commonly used in recombinant microbialexpression vectors include the beta-lactamase (penicillinase) andlactose promoter systems (Chang et al., Nature 275, 615 (1978); andGoeddel et al., Nature 281, 544 (1979)), a tryptophan (trp) promotersystem (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and EPO App.Publ. No. 36,776) and the tac promoter (H. De Boer et al., Proc. Natl.Acad. Sci. USA 80, 21 (1983)). While these are commonly used, othermicrobial promoters are suitable. Details concerning nucleotidesequences of many have been published, enabling a skilled worker tooperably ligate them to DNA encoding the protein in plasmid or viralvectors (Siebenlist et al., Cell 20, 269 (1980)). The promoter andShine-Dalgarno sequence (for prokaryotic host expression) are operablylinked to the DNA encoding the desired protein, i.e., they arepositioned so as to promote transcription of the protein messenger RNAfrom the DNA.

Eukaryotic microbes such as yeast cultures may be transformed withsuitable protein-encoding vectors. See, e.g., U.S. Pat. No. 4,745,057.Saccharomyces cerevisiae is the most commonly used among lowereukaryotic host microorganisms, although a number of other strains arecommonly available. Yeast vectors may contain an origin of replicationfrom the 2 micron yeast plasmid or an autonomously replicating sequence(ARS), a promoter, DNA encoding the desired protein, sequences forpolyadenylation and transcription termination, and a selection gene. Anexemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979);Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157(1980)). This plasmid contains the trp1 gene, which provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12(1977)). The presence of the trpl lesion in the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Publn. No. 73,657.

Cultures of cells derived from multicellular organisms are a desirablehost for recombinant protein synthesis. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture, including insect cells. Propagation of such cellsin cell culture has become a routine procedure. See Tissue Culture,Academic Press, Kruse and Patterson, editors (1973). Examples of usefulhost cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO)cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expressionvectors for such cells ordinarily include (if necessary) an origin ofreplication, a promoter located upstream from the gene to be expressed,along with a ribosome binding site, RNA splice site (ifintron-containing genomic DNA is used), a polyadenylation site, and atranscriptional termination sequence.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells are often providedby viral sources. For example, commonly used promoters are derived frompolyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat.No. 4,599,308. The early and late promoters are useful because both areobtained easily from the virus as a fragment which also contains theSV40 viral origin of replication. See Fiers et al., Nature 273, 113(1978). Further, the protein promoter, control and/or signal sequences,may also be used, provided such control sequences are compatible withthe host cell chosen.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter may besufficient.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperdacells) and expression vectors such as the baculovirus expression vector(e.g., vectors derived from Autographa californica MNPV, Trichoplusia niMNPV, Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed to makeproteins useful in carrying out the present invention, as described inU.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al. In general, abaculovirus expression vector comprises a baculovirus genome containingthe gene to be expressed inserted into the polyhedrin gene at a positionranging from the polyhedrin transcriptional start signal to the ATGstart site and under the transcriptional control of a baculoviruspolyhedrin promoter.

Rather than using vectors which contain viral origins of replication,one can transform mammalian cells by the method of cotransformation witha selectable marker and the chimeric protein DNA. An example of asuitable selectable marker is dihydrofolate reductase (DHFR) orthymidine kinase. See U.S. Pat. No. 4,399,216. Such markers areproteins, generally enzymes, that enable the identification oftransformant cells, i.e., cells which are competent to take up exogenousDNA. Generally, identification is by survival of transformants inculture medium that is toxic, or from which the cells cannot obtaincritical nutrition without having taken up the marker protein.

C. Liqand Analogs and Mimetics

As used herein, "ligand" refers to a molecule that is recognized by aparticular receptor protein. With reference to the present invention, aligand is a molecule, such as a peptide, that is specifically binds toCDK6 at the site bound by the protein having the sequence given hereinas SEQ ID NO:2 and competes with that protein for binding to that site.As used herein, an "inhibitory ligand" or an "inhibitory binding ligand"is a ligand which binds to and inhibits the normal activity of thereceptor protein. "Receptor" refers to a molecule that has an affinityfor a given ligand.

Analogs of p18^(INK6) ligands are an aspect of the present invention. Asused herein, an "analog" is a chemical compound similar in structure toa first compound, and having either a similar or opposite physiologicaction as the first compound. With particular reference to the presentinvention, p18^(INK6) ligand analogs are those compounds which, whilenot having the amino acid sequences of native p18^(INK6) ligands, arecapable of binding to p18^(INK6). Such analogs may be peptide ornon-peptide analogs, including nucleic acid analogs, as described infurther detail below.

In protein molecules which interact with a receptor, the interactionbetween the protein and the receptor must take place atsurface-accessible sites in a stable three-dimensional molecule. Byarranging the critical binding site residues in an appropriateconformation, peptides which mimic the essential surface features of thep18^(INK6) ligands may be designed and synthesized in accordance withknown techniques.

Methods for determining peptide three-dimensional structure and analogsthereto are known, and are sometimes referred to as "rational drugdesign techniques". See, e.g., U.S. Pat. No. 4,833,092 to Geysen; U.S.Pat. No. 4,859,765 to Nestor; U.S. Pat. No. 4,853,871 to Pantoliano;U.S. Pat. No. 4,863,857 to Blalock; (applicants specifically intend thatthe disclosures of all U.S. Patent references cited herein beincorporated by reference herein in their entirety). See also Waldrop,Science, 247, 28029 (1990); Rossmann, Nature, 333, 392-393 (1988); Weiset al., Nature, 333, 426-431 (1988); James et al., Science, 260, 1937(1993) (development of benzodiazepine peptidomimetic compounds based onthe structure and function of tetrapeptide ligands).

In general, those skilled in the art will appreciate that minordeletions or substitutions may be made to the amino acid sequences ofpeptides of the present invention without unduly adversely affecting theactivity thereof. Thus, peptides containing such deletions orsubstitutions are a further aspect of the present invention. In peptidescontaining substitutions or replacements of amino acids, one or moreamino acids of a peptide sequence may be replaced by one or more otheramino acids wherein such replacement does not affect the function ofthat sequence. Such changes can be guided by known similarities betweenamino acids in physical features such as charge density,hydrophobicity/hydrophilicity, size and configuration, so that aminoacids are substituted with other amino acids having essentially the samefunctional properties. For example: Ala may be replaced with Val or Ser;Val may be replaced with Ala, Leu, Met, or Ile, preferably Ala or Leu;Leu may be replaced with Ala, Val or Ile, preferably Val or Ile; Gly maybe replaced with Pro or Cys, preferably Pro; Pro may be replaced withGly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replacedwith Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replacedwith Pro or Cys, preferably Cys; His may be replaced with Phe or Gln,preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferablyHis or Tyr; Tyr may be replaced with His, Phe or Trp, preferably Phe orTrp; Trp may be replaced with Phe or Tyr, preferably Tyr; Asn may bereplaced with Gln or Ser, preferably Gln; Gln may be replaced with His,Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced withGln, Thr, Pro, Cys or Ala; Thr may be replaced with Gln or Ser,preferably Ser; Lys may be replaced with Gln or Arg; Arg may be replacedwith Lys, Asp or Glu, preferably Lys or Asp; Asp may be replaced withLys, Arg, or Glu, preferably Arg or Glu; and Glu may be replaced withArg or Asp, preferably Asp. Once made, changes can be routinely screenedto determine their effects on function with enzymes.

Non-peptide mimetics of the peptides of the present invention are alsoan aspect of this invention. Non-protein drug design may be carried outusing computer graphic modeling to design non-peptide, organic moleculeswhich bind to sites bound by p18^(INK6). See, e.g., Knight,BIO/Technology, 8, 105 (1990). Itzstein et al, Nature, 363, 418 (1993)(peptidomimetic inhibitors of influenza virus enzyme, sialidase).Itzstein et al modeled the crystal structure of the sialidase receptorprotein using data from x-ray crystallography studies and developed aninhibitor that would attach to active sites of the model; the use ofnuclear magnetic resonance (NMR) data for modeling is also known in theart. See also Lam et al, Science, 263, 380 (January 1994) regarding therational design of bioavailable nonpeptide cyclic ureas that function asHIV protease inhibitors. Lam et al used information from x-ray crystalstructure studies of HIV protease inhibitor complexes to designnonpeptide inhibitors.

The modeling of a protein kinase structure using the known structure ofother kinases is reported by Knighton et al., Science, 258, 130 (1992)(smooth muscle myosin light chain kinase catalytic core modeled usingcrystallography data of cyclic AMP-dependent protein kinase catalyticsubunit and a bound pseudosubstrate inhibitor). See also Marcote et al.,Mol. Cell. Biol., 13, 5122 (1993) (crystallography data of cyclic AMPdependent protein kinase used to model Cdc2 protein kinase); Knighton etal., Science, 253, 407 (1991); Knighton et al., Science, 253, 414(1991); DeBondt et al., Nature, 363, 595 (1993) (crystal structure ofhuman CDK2 kinase determined).

Analogs may also be developed by generating a library of molecules,selecting for those molecules which act as ligands for a specifiedtarget, and identifying and amplifying the selected ligands. See, e.g.,Kohl et al., Science, 260, 1934 (1993) (synthesis and screening oftetrapeptides for inhibitors of farnesyl protein transferase, to inhibitras oncoprotein dependent cell transformation). Techniques forconstructing and screening combinatorial libraries of oligomericbiomolecules to identify those that specifically bind to a givenreceptor protein are known. Suitable oligomers include peptides,oligonucleotides, carbohydrates, nonoligonucleotides (e.g.,phosphorothioate oligonucleotides; see Chem. and Engineering News, page20, Feb. 7, 1994) and nonpeptide polymers (see, e.g., "peptoids" ofSimon et al., Proc. Natl. Acad. Sci. USA, 89, 9367 (1992)). See alsoU.S. Pat. No. 5,270,170 to Schatz; Scott and Smith, Science, 249,386-390 (1990); Devlin et al., Science 249, 404-406 (1990); Edgington,BIO/Technology, 11, 285 (1993). Peptide libraries may be synthesized onsolid supports, or expressed on the surface of bacteriophage viruses(phage display libraries). Known screening methods may be used by thoseskilled in the art to screen combinatorial libraries to identify ligandanalogs of p18^(INK6). Techniques are known in the art for screeningsynthesized molecules to select those with the desired activity, and forlabelling the members of the library so that selected active moleculesmay be identified. See, e.g., Brenner and Lerner, Proc. Natl. Acad. Sci.USA, 89, 5381 (1992) (use of genetic tag to label molecules in acombinatorial library); PCT US93/06948 to Berger et al., (use ofrecombinant cell transformed with viral transactivating element toscreen for potential antiviral molecules able to inhibit initiation ofviral transcription); Simon et al., Proc. Natl. Acad. Sci. USA, 89,9367, (1992) (generation and screening of "peptoids", oligomericN-substituted glycines, to identify ligands for biological receptors);U.S. Pat. No. 5,283,173 to Fields et al., (use of genetically alteredSaccharomyces cerevisiae to screen peptides for interactions).

As used herein, "combinatorial library" refers to collections of diverseoligomeric biomolecules of differing sequence, which can be screenedsimultaneously for activity as a ligand for a particular target.Combinatorial libraries may also be referred to as "shape libraries",i.e., a population of randomized polymers which are potential ligands.The shape of a molecule refers to those features of a molecule thatgovern its interactions with other molecules, including Van der Waals,hydrophobic, electrostatic and dynamic.

Nucleic acid molecules may also act as ligands for receptor proteins.See, e.g., Edgington, BIO/Technology, 11, 285 (1993). U.S. Pat. No.5,270,163 to Gold and Tuerk describes a method for identifying nucleicacid ligands for a given target molecule by selecting from a library ofRNA molecules with randomized sequences those molecules that bindspecifically to the target molecule. A method for the in vitro selectionof RNA molecules immunologically cross-reactive with a specific peptideis disclosed in Tsai, Kenan and Keene, Proc. Natl. Acad. Sci. USA, 89,8864 (1992) and Tsai and Keene, J. Immunology, 150, 1137 (1993). In themethod, an antiserum raised against a peptide is used to select RNAmolecules from a library of RNA molecules; selected RNA molecules andthe peptide compete for antibody binding, indicating that the RNAepitope functions as a specific inhibitor of the antibody-antigeninteraction.

C. Use of the Receptors and Proteins

As noted above, the present invention provides isolated and purifiedp18^(INK6) receptor proteins, such as mammalian (or more preferablyhuman) p18^(INK6) receptor proteins. Such proteins can be purified fromhost cells which express the same, in accordance with known techniques,or even manufactured synthetically.

DNAs of the present invention, constructs containing the same and hostcells that express the encoded proteins are useful for making proteinsof the present invention.

Proteins of the present invention are useful as immunogens for makingantibodies as described herein, and these antibodies and proteinsprovide a "specific binding pair." Such specific binding pairs areuseful as components of a variety of immunoassays and purificationtechniques, as is known in the art.

The proteins of the present invention are of known amino acid sequenceas disclosed herein, and hence are useful as molecular weight markers indetermining the molecular weights of proteins of unknown structure.

The DNAs, proteins and mimetics of the present invention can be used ina similar manner as the DNA and proteins of U.S. Pat. No. 5,302,706 toSmith, the disclosure of which is incorporated herein by reference inits entirety.

Antibodies

Antibodies which specifically bind to the proteins of the presentinvention (i.e., antibodies which bind to a single antigenic site orepitope on the proteins) are useful for a variety of diagnosticpurposes. Such antibodies may be polyclonal or monoclonal in origin, butare preferably of monoclonal origin. The antibodies are preferably IgGantibodies of any suitable species, such as rat, rabbit, or horse, butare generally of mammalian origin. Fragments of IgG antibodies whichretain the ability to specifically bind the proteins of the presentinvention, such as F(ab')₂, F(ab'), and Fab fragments, are intended tobe encompassed by the term "antibody" herein. See generally E. Harlowand D. Lane, Antibodies: A Laboratory Manual (1988)(New York: ColdSpring Harbor Laboratory Press). The antibodies may be chimeric, asdescribed by M. Walker et al., Molecular Immunol. 26, 403 (1989).

Monoclonal antibodies which bind to proteins of the present inventionare made by culturing a cell or cell line capable of producing theantibody under conditions suitable for the production of the antibody(e.g., by maintaining the cell line in HAT media), and then collectingthe antibody from the culture (e.g., by precipitation, ion exchangechromatography, affinity chromatography, or the like). The antibodiesmay be generated in a hybridoma cell line in the widely used proceduredescribed by G. Kohler and C. Milstein, Nature 256, 495 (1975), or maybe generated with a recombinant vector in a suitable host cell such asEscherichia coli in the manner described by W. Huse et al., Science 246,1275 (1989).

Immunoassays

Assays for detecting expression of proteins of the present invention ina cell or the extent of expression thereof generally comprise the stepsof, first, contacting cells or extracts of cells to antibodies capableof specifically binding the proteins, and determining the extent ofbinding of said antibodies to said cells. The antibody is preferablylabelled, as discussed above, to facilitate the detection of binding.Any suitable immunoassay procedure may be employed, such asradioimmunoassay, immunofluorescence, precipitation, agglutination,complement fixation, and enzyme-linked immunosorbent assay. When thecells to be tested remain within the body of a mammal, the antibodiesare labelled with a radioactive detectable group and administered to themammal, and the extent of binding of the antibodies to the cells isobserved by external scanning for radioactivity. As discussed above,while any type of antibody may be employed for the foregoing diagnosticpurposes, monoclonal antibodies are preferred. Those skilled in the artwill be familiar with numerous specific immunoassay formats andvariations thereof which may be useful for carrying out the methoddisclosed herein. See generally E Harlow and D. Lane, supra; E. Maggio,Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, FL); see alsoU.S. Pat. No. 4,727,022 to Skold et al.; U.S. Pat. No. 4,659,678 toForrest et al., U.S. Pat. No. 4,376,110 to David et ai., U.S. Pat. No.4,275,149 to Litman et al., U.S. Pat. No. 4,233,402 to Maggio et al.,and U.S. Pat. No. 4,230,767 to Boguslaski et al.

Antibodies may be conjugated to a solid support suitable for adiagnostic assay (e.g., beads, plates, slides or wells formed frommaterials such as latex or polystyrene) in accordance with knowntechniques, such as precipitation. Antibodies may likewise be conjugatedto detectable groups such as radiolabels (e.g., ³⁵ S, ¹²⁵ I, ¹³¹ I),enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), andfluorescent labels (e.g., fluorescein) in accordance with knowntechniques.

Kits for determining if a sample contains proteins of the presentinvention will include at least one reagent specific for detecting thepresence or absence of the protein. Diagnostic kits for carrying outantibody assays may be produced in a number of ways. In one embodiment,the diagnostic kit comprises (a) an antibody which binds proteins of thepresent invention conjugated to a solid support and (b) a secondantibody which binds proteins of the present invention conjugated to adetectable group. The reagents may also include ancillary agents such asbuffering agents and protein stabilizing agents, e.g., polysaccharidesand the like. The diagnostic kit may further include, where necessary,other members of the signal-producing system of which system thedetectable group is a member (e.g., enzyme substrates), agents forreducing background interference in a test, control reagents, apparatusfor conducting a test, and the like. A second embodiment of a test kitcomprises (a) an antibody as above, and (b) a specific binding partnerfor the antibody conjugated to a detectable group. Ancillary agents asdescribed above may likewise be included. The test kit may be packagedin any suitable manner, typically with all elements in a singlecontainer along with a sheet of printed instructions for carrying outthe test.

Nucleic Acid Assays

Assays for detecting p18^(INK6) DNA or mRNA in a cell, or the extent ofamplification thereof, typically involve, first, contacting the cells orextracts of the cells containing nucleic acids therefrom with anoligonucleotide that specifically binds to p18^(INK6) DNA or mRNA asgiven herein (typically under conditions that permit access of theoligonucleotide to intracellular material), and then detecting thepresence or absence of binding of the oligonucleotide thereto. Again,any suitable assay format may be employed (see, e.g., U.S. Pat. No.4,358,535 to Falkow et al.; U.S. Pat. No. 4,302,204 to Wahl et al.;4,994,373 to Stavrianopoulos et al; 4,486,539 to Ranki et al.; 4,563,419to Ranki et al.; and 4,868,104 to Kurn et al.)(the disclosures of whichapplicant specifically intends be incorporated herein by reference).

Antisense Oligonucleotides

Antisense oligonucleotides and nucleic acids that express the same maybe made in accordance with conventional techniques. See, e.g., U.S. Pat.No. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al. Thelength of the antisense oligonucleotide (i.e., the number of nucleotidestherein) is not critical so long as it binds selectively to the intendedlocation, and can be determined in accordance with routine procedures.In general, the antisense oligonucleotide will be from 8, 10 or 12nucleotides in length up to 20, 30, or 50 nucleotides in length. Suchantisense oligonucleotides may be oligonucleotides wherein at least one,or all, of the internucleotide bridging phosphate residues are modifiedphosphates, such as methyl phosphonates, methyl phosphonothioates,phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Forexample, every other one of the internucleotide bridging phosphateresidues may be modified as described. In another non-limiting example,such antisense oligonucleotides are oligonucleotides wherein at leastone, or all, of the nucleotides contain a 2' loweralkyl moiety (e.g.,C1-C4, linear or branched, saturated or unsaturated alkyl, such asmethyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl).For example, every other one of the nucleotides may be modified asdescribed. See also P. Furdon et al., Nucleic Acids Res. 17, 9193-9204(1989); S. Agrawal et al., Proc. Natl. Acad. Sci. USA 87, 1401-1405(1990); C. Baker et al., Nucleic Acids Res. 18, 3537-3543 (1990); B.Sproat et al., Nucleic Acids Res. 17, 3373-3386 (1989); R. Walder and J.Walder, Proc. Natl. Acad. Sci. USA 85, 5011-5015 (1988).

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In these examples, Dmeans Dalton, kD means kilodalton, mM means milliMolar, and temperaturesare given in degrees Centigrade unless otherwise indicated.

EXAMPLE 1 Isolation of p18^(INK6)

Human p18^(INK6) was isolated by two-hybrid screening (Fields and Song,Nature 340:245-246 (1989) as follows. The complete open reading frame ofhuman cyclin-dependent kinase 6 (CDK6; Meyerson et al., EMBO J., 11,2909-2917 (1992)) was inserted into the vector pGBT8 (constructed by Y.Xiong), a modified form of the yeast two hybrid screening vector pGBT9(constructed by S. Field) which directs the expression of a fusionbetween the DNA-binding domain (amino acids 1-147) of Gal-4 and theentire CDK6 protein from a crippled ADH promoter. This plasmid, whichalso contains a TRP1 marker, was co-transformed into yeast strain Y190(W. Harper et al., Cell 75, 805-816 (1993)) with the human HeLa cDNAlibrary constructed in vector pGADGL (Clontech Inc.) that carries a Leumarker. Transformants were plated on yeast drop-out media lackingleucine, tryptophan, and histidine, and containing 30 mM 3-amino-l,2,4triazole (3-AT). An estimated 5×10⁶ transformants were screened. After 6days of growth, histidine positive (His+) colonies were tested forb-galactosidase activity. Forty-five colonies were positive forb-galactosidase staining and were further purified on selective media.Plasmid DNA was recovered from positive colonies and introduced intoEscherichia coli strain JM101 (J. Messing, Recomb. DNA Tech. bull. 2(2): 43 (1979)) .

The cDNAs from plasmids recovered from E. coli were excised from pGADGLvector and inserted into pBluescript (Stratagene Inc.). The first threehundred nucleotide sequences were determined for each clone. Most clonescorresponded to the previously reported p16^(INK4/Mts1). In addition top16^(INK4), a clone (6H10) was isolated that encoded a novel proteinthat was not present in the current data base ((GenBank release 82.00April 1994).

A cDNA insert from clone 6H10 was used as probe to screen a human HeLacDNA library constructed in λZAP II vector (STRATAGENE Inc.) to obtainfull length sequences. More than twenty lambda clones were isolated fromthis screening and several of them were analyzed by DNA sequencing. Oneclone, H18, was determined to contain a full length coding region, asthere was an in-frame stop codon located 6 base-pairs upstream of aputative initiation ATG codon (see SEQ ID NO: 1). The translation ofclone H18 contained 168 amino acid residues with a calculated molecularweight of 18116 D (or 18 kDa, p18) and showed 40% protein sequencesimilarity to p16^(INK4/MTs1). This protein was named p18^(INK6),following the existing nomenclature and because of the preferentialinteraction of p18^(INK6) with CDK6.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 634 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 94..597                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCGATGCCATCATGCAGCCTGGTTAGGAGCAAAGGAAAGGGGAAAAAGAAAAACGACTAA60                TTCATCTTTTCCTGATCGTCAGGACCCTAAAGAATGGCCGAGCCTTGGGGGAAC114                     MetAlaGluProTrpGlyAsn                                                         15                                                                            GAGTTGGCGTCCGCAGCTGCCAGGGGGGACCTAGAGCAACTTACTAGT162                           GluLeuAlaSerAlaAlaAlaArgGlyAspLeuGluGlnLeuThrSer                              101520                                                                        TTGTTGCAAAATAATGTAAACGTCAATGCACAAAATGGATTTGGAAGG210                           LeuLeuGlnAsnAsnValAsnValAsnAlaGlnAsnGlyPheGlyArg                              253035                                                                        ACTGCGCTGCAGGTTATGAAACTTGGAAATCCCGATATTGCCAGGAGA258                           ThrAlaLeuGlnValMetLysLeuGlyAsnProAspIleAlaArgArg                              40455055                                                                      CTGCTACTTAGAGGTGCTAATCCCGATTTGAAAGACCGAACTGGTTTC306                           LeuLeuLeuArgGlyAlaAsnProAspLeuLysAspArgThrGlyPhe                              606570                                                                        GCTGTCATTCATGATGCGGCCAGAGCAGGTTTCCTGGACACTTTACAG354                           AlaValIleHisAspAlaAlaArgAlaGlyPheLeuAspThrLeuGln                              758085                                                                        ACTTTGCTGGAGTTTCAAGCTGATGTTAACATCGAGGATAATGAAGGG402                           ThrLeuLeuGluPheGlnAlaAspValAsnIleGluAspAsnGluGly                              9095100                                                                       AACCTGCCCTTGCACTTGGCTGCCAAAGAAGGCCACCTCCGGGTGGTG450                           AsnLeuProLeuHisLeuAlaAlaLysGluGlyHisLeuArgValVal                              105110115                                                                     GAGTTCCTGGTGAAGCACACGGCCAGCAATGTGGGGCATCGGAACCAT498                           GluPheLeuValLysHisThrAlaSerAsnValGlyHisArgAsnHis                              120125130135                                                                  AAGGGGGACACCGCCTGTGATTTGGCCAGGCTCTATGGGAGGAATGAG546                           LysGlyAspThrAlaCysAspLeuAlaArgLeuTyrGlyArgAsnGlu                              140145150                                                                     GTTGTTAGCCTGATGCAGGCAAACGGGGCTGGGGGAGCCACAAATCTT594                           ValValSerLeuMetGlnAlaAsnGlyAlaGlyGlyAlaThrAsnLeu                              155160165                                                                     CAATAACGTGGGGAGGGCTCCCCCACGTTGCCTCTAAAAA634                                   Gln                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 168 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaGluProTrpGlyAsnGluLeuAlaSerAlaAlaAlaArgGly                              151015                                                                        AspLeuGluGlnLeuThrSerLeuLeuGlnAsnAsnValAsnValAsn                              202530                                                                        AlaGlnAsnGlyPheGlyArgThrAlaLeuGlnValMetLysLeuGly                              354045                                                                        AsnProAspIleAlaArgArgLeuLeuLeuArgGlyAlaAsnProAsp                              505560                                                                        LeuLysAspArgThrGlyPheAlaValIleHisAspAlaAlaArgAla                              65707580                                                                      GlyPheLeuAspThrLeuGlnThrLeuLeuGluPheGlnAlaAspVal                              859095                                                                        AsnIleGluAspAsnGluGlyAsnLeuProLeuHisLeuAlaAlaLys                              100105110                                                                     GluGlyHisLeuArgValValGluPheLeuValLysHisThrAlaSer                              115120125                                                                     AsnValGlyHisArgAsnHisLysGlyAspThrAlaCysAspLeuAla                              130135140                                                                     ArgLeuTyrGlyArgAsnGluValValSerLeuMetGlnAlaAsnGly                              145150155160                                                                  AlaGlyGlyAlaThrAsnLeuGln                                                      165                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TNGCATCNGCAGCAGCACGNGG22                                                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CACGACGCAGCACGNGCAGGNTTCCTNGACACNCT35                                         __________________________________________________________________________

That which is claimed is:
 1. An isolated nucleic acid encoding a CDK6inhibiting protein, said nucleic acid sequence selected from the groupconsisting of:(a) DNA having the nucleotide sequence given herein as SEQID NO:1; and (b) nucleic acids which differ from the nucleic acid of (a)above due to the degeneracy of the genetic code, and which encode theCDK6 inhibiting protein encoded by the DNA of (a) above.
 2. An isolatednucleic acid which encodes a CDK6 inhibiting protein having the aminoacid sequence given herein as SEQ ID NO:2.
 3. An isolated nucleic acidaccording to claim 1 above which is a DNA having the nucleotide sequencegiven herein as SEQ ID NO:1.
 4. A nucleic acid construct having apromoter and a heterologous nucleic acid operably linked to saidpromoter, wherein said heterologous nucleic acid is a nucleic acidaccording to claim 1 or
 2. 5. A cell containing a nucleic acid constructaccording to claim
 4. 6. A cell containing a nucleic acid constructaccording to claim 4 and capable of expressing the encoded protein.